Chen L Q, Santarelli V, Horn R, Kallen R G
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104-6059, USA.
J Gen Physiol. 1996 Dec;108(6):549-56. doi: 10.1085/jgp.108.6.549.
Sodium channels have four homologous domains (D1-D4) each with six putative transmembrane segments (S1-S6). The highly charged S4 segments in each domain are postulated voltage sensors for gating. We made 15 charge-neutralizing or -reversing substitutions in the first or third basic residues (arginine or lysine) by replacement with histidine, glutamine, or glutamate in S4 segments of each domain of the human heart Na+ channel. Nine of the mutations cause shifts in the conductance-voltage (G-V) midpoints, and all but two significantly decrease the voltage dependence of peak Na+ current, consistent with a role of S4 segments in activation. The decreases in voltage dependence of activation were equivalent to a decrease in apparent gating charge of 0.5-2.1 elementary charges (eo) per channel for single charge-neutralizing mutations. Three charge-reversing mutations gave decreases of 1.2-1.9 eo per channel in voltage dependence of activation. The steady-state inactivation (h infinity) curves were fit by single-component Boltzmann functions and show significant decreases in slope for 9 of the 15 mutants and shifts of midpoints in 9 mutants. The voltage dependence of inactivation time constants is markedly decreased by mutations only in S4D4, providing further evidence that this segment plays a unique role in activation-inactivation coupling.
钠通道有四个同源结构域（D1 - D4），每个结构域有六个假定的跨膜片段（S1 - S6）。每个结构域中带高电荷的S4片段被假定为门控的电压传感器。我们通过将人心脏钠通道每个结构域S4片段中的第一个或第三个碱性残基（精氨酸或赖氨酸）替换为组氨酸、谷氨酰胺或谷氨酸，进行了15个电荷中和或反转替代。其中九个突变导致电导 - 电压（G - V）中点发生偏移，除两个突变外，所有突变均显著降低了峰值钠电流的电压依赖性，这与S4片段在激活过程中的作用一致。对于单个电荷中和突变，激活电压依赖性的降低相当于每个通道表观门控电荷减少0.5 - 2.1个基本电荷（eo）。三个电荷反转突变使激活电压依赖性每个通道降低1.2 - 1.9 eo。稳态失活（h无穷大）曲线用单组分玻尔兹曼函数拟合，结果显示15个突变体中有9个的斜率显著降低，9个突变体的中点发生偏移。仅S4D4中的突变显著降低了失活时间常数的电压依赖性，这进一步证明该片段在激活 - 失活偶联中起独特作用。
